Summary of Work: AIMS: Recent studies suggest important roles for glial cells (astrocytes and microglia) in the development, differentiation and survival of neurons in the brain. Neurotoxins released from microglial cells (the main immune cells in the brain) in response to adverse neuroimmune reactions may cause the death of neurons and is believed to be the major cause of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The purpose of this project was to evaluate the roles of opioid peptides, enkephalins (ENK), dynorphins (DYN) and b-endorphin (b-END), in the interactions between glia and neurons. Our major efforts focused on the influences of opioid peptides on the release of proinflammatory cytokines, production of reactive nitrogen free radicals, and neurotoxicities induced by endotoxins or neurotoxins. In order to achieve these goals, we have developed a mixed neuronal/glial cell co-culture system as a model to examine the roles of microglia and astroglia on the survival of neurons in response to various chemical insults. ACCOMPLISHMENTS: One of the most interesting findings from this series of studies was the potent inhibition by DYN of lipopolysaccharide (LPS)-induced effects. Ultra low concentrations of DYN (10-14 to 10-16 M) were found to inhibit the LPS-induced release of cytokines and nitric oxide in mixed glia cell cultures. Further study revealed that the inhibitory effect of DYN results from the reduction in LPS-induced activation of microglia. Using mixed neuron/glia cultures as a model of neurotoxicity, we have recently observed that the same ultra low concentrations of DYN reduce LPS-induced neuronal cell death. This in vitro finding was further substantiated when the LPS-induced loss of dopamine-containing neurons in the brain was prevented by infusion of DYN in whole animal studies. Since these ultra low concentrations of DYN are within the range of concentrations in the brain, this finding suggests that DYN is an endogenous immune modulator in the brain. Furthermore, fentomolar concentrations of DYN are well below the dissociation constant (Kd) values for most of the known opiate receptors (Kd around 10-9 M), suggesting that a novel non-opiate receptor mediates this inhibitory action of DYN. Preliminary studies from our laboratory which showed that the inhibitory effects of DYN on the LPS-induced release of TNF-a was not blocked by naloxone and a non-opioid DYN analog, des-[Tyr1]-dynorphin, exhibited the same inhibitory effects as DYN also suggest the involvement on non-opiate receptor mechanisms. Thus, characterization and cloning of this non-opiate receptor from microglial cells should reveal an entirely new class of signal transduction system regulating neuroimmune functions in the brain.